The incidence of Human Papilloma Virus related to head and neck squamous cell carcinoma (HPV+ HNSCC) is rapidly increasing in the United States. HPV+ HNSCC has distinct etiologic, demographic, and clinico- pathologic characteristics in comparison to non-HPV related HNSCC. However, the therapy development for HPV+ HNSCC is challenging due to a low incidence of targetable oncogenic mutations and limited transcriptional characterization in comparison to other solid tumors. The investigation of alternative RNA splicing events (ASEs), provides insight into novel transcription alterations, and can expand the transcriptional characterization of HPV+ HNSCC. Our project will define HPV+ HNSCC specific alternative splicing events via two specific aims: (1) Definition of differential alternative splicing in primary HPV+ HNSCC, and (2) Validation of differential alternative splicing in HPV+ HNSCC. We will use outlier analysis in combination with high throughput analysis of transcriptional products to define high value ASEs specific for HPV+ HNSCC. We will perform validation of detected ASE candidates using multiple cohort and complementary validation techniques. This project will provide insight into HPV+ HNSCC biology and will potentially provide therapeutic and prognostic targets for the development of HPV+ HNSCC specific therapies. The data developed during the proposed R21 award will be used as preliminary data for an R01 grant, seeking to discover the functional role of splicing events and correlation of alternative splicing with patient clinicopathological parameters in order to develop the translational therapeutic and clinical application of targeting HPV+ HNSCC specific ASEs.